How to grow more Agrobacterium? - (Sep/15/2005 )

We found a strain of bacterium from deep rocks.

It probably is Agrobacterium tumefaciens.(98% identity in 16s RNA)

I knew that A. tumefaciens is a useful tool to transfer genes into plant so I want to ask if anyone know how to grow them better? The amount of my bacteria is poor

3q

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We have no problems growing large amounts of Agrobacterium in our lab using liquid YEP medium (1% yeast extract, 1% tryptone peptone, 0.5% NaCl, pH 7.0) at 27 degrees and shaking at about 200 rpm. Normally we see growth within a day or two and the cultures end up with a large amount of bacteria. This media also works well in plates.

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I agree, usually it's very simple to grow large amount of Agrobacterium.
Another way to grow it is using LB Miller medium, changing a bit its composition according to the strain used.
The basic formula is: 10g/l tryptone, 5g/l yeast extract, 5g/l NaCl, but sometimes we add also 1g/l glucose and/or vary NaCl amount from 5 to max. 10g/l.
If you want to try with solid medium, you have to add 15g/l agar or bactoagar.

Hope this could help!

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I have been used these medium but It seem not perfect.
Some time they don't grow on solid LB, I don't know why? May be it depend on my genus I used
If anybody invent a medium that we can grow Agro like E.coly ...
good for us but the GMO analysizer will kill him immediatelly

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At which temperature are you trying to grow agro? We usually grow them ~30C instead of 37 as 37 tended to either stop ours from growing or killed them outright. I am not sure if anyone else has had this experience?

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...different habits from different labs: we are used to grow it at 32°C, 150rpm!!!

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oh we grow for competent cells around 30 , in LB medium witout antibiotics
but after transformation only at 27-28 in LB + antibiotics

i read in a book that after 30 degree the agrobacterium loses its plasmid


any truth ??


laxmi

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hi

faster but do they keep their plasmids ??

plus we use C58 strain so may be different

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We grow LBA4404 and EHA105 engineered strains to perform genetic transformation of plant.
I think we have never observed any plasmid loss...but I would really like to check deeper this information. Do you remember your source book?
Thanks in advance!

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hi

it was one year back that i saw it in lib , i think
it was a called "Agrobacterium Protocols: Volume II (Methods in Molecular Biology) "
by Kan Wang (Editor)

blue cover ,
second edition i think

i got it from the lib and returned it , a long way back so sorry for the memory loss
if i see it again and if there is anything else to add i will send it to u

regards
laxmi

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